The invention relates to compositions of poorly water-soluble drugs such as triterpenes and methods for preparing the compositions. Particularly, the invention relates to compositions comprising pentacyclic triterpenes and more particularly, betulinic acid and its derivatives. The present invention also relates to betulinic acid and/or its derivatives that are used for the treatment and suppression of malignant diseases including but not limited to leukemias, lymphomas, melanoma, prostate and ovarian cancers.
Under the auspices of a National Cooperative Natural Product Drug Discovery Group supported by the National Cancer Institute, the potential anti-tumor activity of approximately 2500 extracts derived from globally collected plants were evaluated in a panel of enzyme based assays and in a battery of cultured human tumor cell lines. One such extract, prepared from the stem bark of Ziziphus mauritiana Lam. (Rhamnaceae) displayed selective cytotoxicity against cultured human melanoma cells (Nature Medicine, Vol. 1 (10) 1995, WO 96/29068). As a result of bioactivity guided fractionation, betulinic acid, a pentacyclic triterpene, was identified as a melanoma-specific cytotoxic agent. In follow-up studies conducted with athymic mice carrying human melanomas, tumor growth was completely inhibited without toxicity. As judged by a variety of cellular responses, anti-tumor activity was mediated by the induction of apoptosis.
A number of triterpenoids, including betulinic acid, have several known medical applications, including use as an anticancer drug. Anderson et al., in WO 95/04526, discuss derivatives of triterpenoids which have been used in cancer therapy, including their activity against polyamines which are required by cells to grow at an optimal rate. Some of these triterpenoids have been found to interfere with enzymatic synthesis of polyamines required for optimal cell growth, and thus inhibit the growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K. et al. Oncology 48:72-76, 1991). The anti-cancer activity of betulinic acid and some derivatives has been demonstrated using mouse sarcoma 180 cells implanted subcutaneously in nude mice (JP 87,301,580). Choi et al have shown that betulinic acid 3-monoacetate, and betulinic acid methyl ester exhibit ED50 values of 10.5 and 6.8 xcexcg/ml, respectively against P388 lymphocytic leukemia cells (Choi, Y. H. et al. Planta Medica vol. XLVII, pages 511-513, 1988). Betulinic acid and derivatives of betulinic acid have highly selective activity against melanoma cells, murine carcinosarcoma and murine lymphocytic leukemia.
The selective cytotoxicity of betulinic acid and its various derivatives, and their lack of toxicity towards normal cells, afford a favorable therapeutic index. However, the poor solubility of betulinic acid and its derivatives has limited research on other activities of betulinic acid and its derivatives. This is also reflected by the fact that except for a few topical preparations no systemic preparations for the administration of betulinic acid and/or its derivatives are reported. This is due to very poor aqueous solubility of the betulinic acid and its derivatives. To date there is no formulation reported for the administration of these drugs for human use.
There is a need, therefore, for solubilization of betulinic acid and/or its derivatives into pharmaceutically acceptable compositions which are miscible with aqueous intravenous diluting fluids.
It is an important objective of the present invention to solubilize derivatives of betulinic acid and/or its derivatives that have limited utility due to poor solubility.
It is another important objective of the present invention to overcome the poor solubility of the betulinic acid and its derivatives by solubilizing them in a pharmaceutically acceptable non-toxic solvent system.
Another very important objective of the present invention is to provide a pharmaceutically acceptable composition of betulinic acid and its derivatives that can be utilized for the treatment of malignant diseases.
The present invention aims to overcome the above problems and realize the objects of the present invention by providing a novel, pharmaceutically acceptable solvent system based, inter alia, upon the principles of co-solvency. The solvent system comprises an organic solvent, a co-solvent and a solubilizer.
The present invention provides compositions and methods for the solubilization of poorly soluble drugs such as triterpenes like betulinic acid and/its derivatives in pharmaceutically acceptable liquid vehicles that avoid use of potentially toxic solvents that are often used for the solubilization of poorly soluble drugs. In the compositions of this invention the drugs remain physically and chemically stable and can be administered intravascularly without undue toxicity from undissolved drug and/or from the solvent vehicles at a drug dose meant to be effective to exhibit clinically significant anticancer activity. Before intravascular administration, the compositions are diluted in a pharmaceutically acceptable solution that is suitable for intravascular administration.
According to the present invention, a triterpene is dissolved in an organic solvent, followed by the addition of a co-solvent and a solubilizer. Examples of organic solvents that can be used are dimethylacetamide (DMA), dimethylsulphoxide (DMSO), and alcohols such as methanol, ethanol, propanol and isopropanol. Examples of co-solvents that can be used are polyethylene glycol (PEG), ethanol, various amides such as pyrrolidinone or 1-methyl-2-pyrrolidinone, or sulfur containing compounds such as sulfolane, dimethylsulfoxide (DMSO) or tetramethylene sulfoxide. Any other co-solvent that would be suitable for use in this invention can also be used. Preferably, polyethylene glycol (PEG) is used as the co-solvent. The molecular weight of the PEG may vary from about 300 to about 10,000. More preferably the PEG has an average molecular weight of about 300. Examples of solubilizers that can be used include polyoxyethylene sorbitan fatty acid esters, poloxamers, polyoxyethylene stearates or lecithin. A polyoxyethylene sorbitan fatty acid ester such as POLYSORBATE 20, 21, 40, 60, 61, 65, 80, 81, 85 or 120, or TWEEN 80, preferably POLYSORBATE 80 or TWEEN 80, a poloxamer such as a PLURONIC or a polyoxyethylene stearate such as MYRJ 52 can be used as the solubilizer. Any other solubilizer that would be suitable for use in this invention can be used.
According to the invention, betulinic acid or any of its derivatives is dissolved in an organic solvent as the primary vehicle, for example, dimethylacetamide (DMA), followed by the addition of co-solvent(s) such as polyethylene glycol and a solubizer such as polyoxyethylene sorbitan fatty acid ester to retain the drug in solution upon dilution with an aqueous vehicle.
The preferred compositions utilize combinations of anhydrous dimethylacetamide (DMA), as the primary solvent, polyethylene glycol 300 (PEG) as the co-solvent and a polyoxyethylene sorbitan fatty acid ester such as POLYSORBATE 80 as the solubilizer. Such compositions are miscible in solutions that are used in intravenous preparations. These intravenous solutions include but are not limited to solutions that contain 2-25% by weight of dextrose such as 5% dextrose solution, 10% dextrose solution, normal saline or dextrose-normal saline. Any intravenous solution that can be used for humans or animals can be used. These intravenous solutions are examples of vehicles in which betulinic acid or its derivatives are effectively solubilized and can be administered to humans or animals, alone or in combination with other drugs.
Accordingly, the present invention provides a novel pharmaceutical composition for solubilization of triterpenes which comprises from 5% to 50% by volume of an organic solvent, from 20% to 80% by volume of a co-solvent and from 5% to 60% by weight of a solubilizer. Optionally, the composition is diluted with an aqueous intravenous diluting fluid.
In one embodiment of the invention the composition for solubilization of triterpenes comprises from 5-50% of dimethylacetamide (DMA), from 20-80% by volume of polyethylene glycol (PEG) and from 5-60% by weight of polyoxyethylene sorbitan fatty acid ester such as Tween.
In a preferred embodiment, the pharmaceutical composition for solubilization of triterpenes comprises from 20% to 50% by volume of dimethylacetamide (DMA), from 20% to 40% by volume of polyethylene glycol (PEG) and from 5% to 30% by weight of polyoxyethylene sorbitan fatty acid ester such as Tween. Optionally, the composition is diluted with an aqueous intravenous diluting fluid.
In a still more preferred embodiment, the DMA and polyoxyethylene sorbitan fatty acid ester such as TWEEN 80 are present at a ratio of about 1:2 (v/v) and in another preferred embodiment, the DMA and PEG 300 are present at a ratio of about 1:4 (v/v).
It will be understood by a skilled artisan that the present invention will work even outside these ratios.
Preferably the triterpene is betulinic acid 
or a derivative thereof.
Given below is the list of some of the representative derivatives of betulinic acid that can be solubilized using the above compositions.
Wherein, Betulinic acid derivatives representing R, R1, R2, R3, and R4 are selected from the following as shown in the Table herein below:
In a preferred embodiment the concentration of triterpene is about 0.001 to 40 mg/ml of the composition before it is diluted with an intravenous solution.
The present invention also provides a method for solubilizing triterpenes which comprises the steps of:
dissolving said triterpene in from 5% to 50% by volume of an organic solvent, from 20% to 80% by volume of a co-solvent and from 5% to 60% by weight of a solubilizer and if desired, diluting said composition with an aqueous intravenous diluting fluid.
The present invention also provides a method for solubilizing triterpenes which comprises the steps of:
dissolving said triterpene in from 5% to 50% by volume of dimethylacetamide (DMA), from 20% to 80% by volume of polyethylene glycol (PEG) and from 5% to 60% by weight of polyoxyethylene sorbitan fatty acid ester and if desired, diluting said composition with an aqueous intravenous diluting fluid.
Preferably, said triterpenes are selected from betulinic acid and its derivatives. More preferably, PEG is added to the solution of betulinic acid or its derivatives at a ratio of DMA:PEG of about 1:4 (v/v), followed by the addition of a polyoxyethylene sorbitan fatty acid ester such as TWEEN 80 at a ratio of DMA: polyoxyethylene sorbitan fatty acid ester of about 1:2 (v/v) to obtain a concentration of betulinic acid or its derivatives of about 0.001 mg/ml to 40 mg/ml. The solution can be diluted to a concentration of triterpene between 0.0001 to less than 40 mg/ml.
The present invention will now be described with reference to the following non-limiting examples.